Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.
Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.
Li
J
J
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038, USA.
Gao
E
E
Mendelson
C R
CR
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010215:Papio; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011510:Proteolipids / Q000235:genetics*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C445843:SFTPA2 protein, human; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D003994:Bucladesine; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate